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死亡结构域相关蛋白Daxx可以敏化多种肿瘤细胞的凋亡过程,但对于肝肿瘤细胞株HepG2的影响未见报道.为了研究Daxx增加肝HepG2细胞对药物敏感性的影响及机制,为开发药物新的药理作用提供理论依据,分别转染pEGFP-C1和pEGFP-C1-Daxx这两个载体到HepG2细胞.实验分组如下:(1)正常对照组(未转染细胞组);(2)pEGFP-C1空载体转染组(HepG2/GFP细胞);(3)pEGFP-C1-Daxx表达载体转染组(HepG2/GFP-Daxx细胞).筛选稳定细胞株,用逆转录聚合酶链反应检测mRNA的表达;用过氧化氢孵育24h诱导细胞凋亡,采用MTT法和流式细胞术检测细胞凋亡率,Western blot检测蛋白质的表达.经G418筛选稳定的细胞运用RT-PCR技术分析其mRNA,结果显示,转染绿色荧光蛋白Daxx表达载体的细胞Daxx的mRNA明显上调;用荧光显微镜观察到Daxx蛋白主要定位于细胞核.用过氧化氢诱导HepG2细胞凋亡,观察到过氧化氢呈浓度依赖性地抑制HepG2细胞活性.正常对照细胞、HepG2/GFP、HepG2/GFP-Daxx 3组细胞的IC50值分别是0.72、0.76、0.49mmol/L.并且运用流式细胞仪检测到HepG2/GFP-Daxx组细胞凋亡率明显高于转染空载体质粒组与未转染组((42.9±8.42)vs(27.3±6.38)or(28.5±4.71)).提示HepG2/GFP-Daxx细胞对过氧化氢的反应性较未转染细胞和HepG2/GFP敏感.还运用Western-blot检测到活化的caspase3在Daxx转染组细胞表达最强,达到(204.66±19.68)%,而未转染和HepG2/GFP组细胞分别是(100±3.1)%、(107.39±20.1)%,进一步说明了Daxx可以增加HepG2细胞对于过氧化氢的敏感性.同时,观察到过氧化氢处理24h后,Daxx转染组细胞磷酸化的JNK表达明显高于空载体转染组和未转染细胞组.上述结果表明:a.Daxx可以增加肝HepG2细胞对过氧化氢诱导的细胞凋亡敏感性;b.Daxx蛋白敏化过氧化氢诱导的HepG2细胞凋亡可能与协同增加JNK活性有关.
The death domain-related protein Daxx can sensitize the apoptosis process of many kinds of tumor cells, but the effect on HepG2 has not been reported.In order to study the effect and mechanism of Daxx on the drug sensitivity of HepG2 cells, The new pharmacological effects provide theoretical basis for the transfection of two vectors pEGFP-C1 and pEGFP-C1-Daxx to HepG2 cells.The experimental groups are as follows: (1) normal control group (untransfected cells group); (2) pEGFP (HepG2 / GFP cells); (3) HepG2 / GFP-Daxx cells transfected with pEGFP-C1-Daxx expression vector. Stable cell lines were screened and mRNA was detected by reverse transcription polymerase chain reaction The cells were incubated with hydrogen peroxide for 24 hours to induce cell apoptosis.MTT assay and flow cytometry were used to detect the apoptosis rate.Western blot was used to detect the protein expression.The stable cells were screened by G418 and analyzed by RT- The results showed that Daxx mRNA was up-regulated in Daxx expression vector transfected with green fluorescent protein Daxx, and Daxx protein mainly localized in nucleus with fluorescence microscope.Hydrogen peroxide induced apoptosis of HepG2 cells in a dose-dependent manner Inhibit HepG2 cell viability The IC50 values of normal control cells, HepG2 / GFP and HepG2 / GFP-Daxx 3 groups were 0.72,0.76,0.49mmol / L. And the apoptosis rate of HepG2 / GFP-Daxx group was detected by flow cytometry (42.9 ± 8.42) vs (27.3 ± 6.38) or (28.5 ± 4.71), respectively, which indicated that the reactivity of HepG2 / GFP-Daxx cells to hydrogen peroxide was not as high as that of the untransfected cells Transfected cells and HepG2 / GFP were sensitive.Western blot also showed that activated caspase3 was the strongest in Daxx transfection group (204.66 ± 19.68%), while cells in untransfected and HepG2 / GFP groups were ( 100 ± 3.1)%, (107.39 ± 20.1)%, which further demonstrated that Daxx could increase the sensitivity of HepG2 cells to hydrogen peroxide.At the same time, the phosphorylation of JNK in Daxx transfection group was observed after 24 h of hydrogen peroxide treatment Was significantly higher than that in the empty vector transfected and untransfected cells.The above results show that: a.Daxx can increase the hepatoma HepG2 cell sensitivity to hydrogen peroxide-induced apoptosis; b.Daxx protein sensitized to hydrogen peroxide-induced HepG2 cell apoptosis may be related to the synergistic increase of JNK activity.